The invention concerns a means for delivering to a patient a therapeutically effective amount of an integrin receptor antagonist such as a fibrinogen receptor (.alpha..sub.II .beta..sub.3, also referred to as GP IIb/IIIa) antagonist or a vitronectin receptor (.alpha..sub.v .beta..sub.3) antagonist.
Integrins constitute an extended family ("superfamily") of membrane receptors interacting with adhesive proteins in plasma and extracellular matrix and with other membrane receptors (counter-receptors). The name "integrin" implies that they integrate the ligands on the outside of the cell with the cytoskeletal apparatus in the inside of the cell. Integrin receptors consist of a noncovalently lined Ca.sup.2+ -dependent, heterodimeric glycoprotein complex composed of .alpha. and .beta. subunits.
The eight known integrin .alpha. subunits give rise to eight families in which one "founder" .beta. subunit forms heterodimers with different .alpha. subunits. There are at least 14 known .alpha. subunits. Among them .alpha..sub.v ("v" stands for association with the vitronectin receptor) seems to be most promiscuous, forming liaisons with six different .beta. subunits. Receptors belonging to the .beta..sub.1 and .beta..sub.3 families are expressed in endothelial cells. The .beta..sub.1 family, also named Very Late Antigens (VLA), is represented by the fibronectin receptor (.alpha..sub.5 .beta..sub.1, or VLA-5), the collagen receptor (.alpha..sub.2 .beta..sub.1, or VLA-2) and the laminin receptor (.alpha..sub.6 .beta..sub.1). The .beta..sub.3 family is represented by the vitronectin receptor (.alpha..sub.v .beta..sub.3), which is structurally similar (the same .beta..sub.3 subunit) to the platelet integrin receptor for fibrinogen, glycoprotein IIb-IIIa complex (.alpha..sub.IIb .beta..sub.3). The functional difference between these two receptors is that the platelet receptor recognizes the .gamma. chain domain (HHLGGAKQAGDV) of human fibrinogen and the endothelial vitronectin receptor does not. Both recognize the sequence R-G-D identified as the cell adhesion site of fibronectin, vitronectin, vWf, and the .alpha. chain of human fibrinogen. Therefore, synthetic peptides containing the R-G-D sequence cause detachment of endothelial cells from the extracellular in matrix in vitro.
The final obligatory step in platelet aggregation is the binding of fibrinogen to an activated membrane-bound glycoprotein complex, GP IIb/IIIa (.alpha..sub.II .beta..sub.3). Platelet activators such as thrombin, collagen, epinephrine or ADP, are generated as an outgrowth of tissue damage. During activation, GP IIb/IIIa undergoes changes in conformation that result in exposure of occult binding sites for fibrinogen. There are six putative recognition sites within fibrinogen for GP IIb/IIIa and thus fibrinogen can potentially act as a hexavalent ligand to crossing GP IIb/IIIa molecules on adjacent platelets. A deficiency in either fibrinogen or GP IIb/IIIa prevents normal platelet aggregation regardless of the agonist used to activate the platelets. Since the binding of fibrinogen to its platelet receptor is an obligatory component of normal aggregation, GP IIb/IIIa is an attractive target for an antithrombotic agent.
The snake venom proteins, termed disintegrins, have provided important structural information for identifying fibrinogen receptor antagonists, but their antigenicity has limited their development as therapeutic agents (Cook et al. ibid.; and Cox et al. ibid.). Integrilin is a cyclic peptide that is based on the KGD sequence in the snake venom protein barbourin (Cook et al. ibid.; and Cox et al. ibid.). It inhibits ligand binding to GPIIa/IIIa but has very little effect on ligand binding to .alpha..sub.v .beta..sub.3. Among the non-peptide compounds are Ro 44-9883 and MK-383, which are administered intravenously, and are also selective for GPIIb/IIIa (Cook et al. ibid.; and Cox et al. ibid.). Orally active agents include SC54684, which is a prodrug (i.e., it requires biotransformation in vivo to its active form) with high oral bioavailability and Ro 43-8857, GR144053, and DMP728, which are themselves the active inhibitors (Cook et al. ibid.; and Cox et al. ibid.). Literally thousands of other compounds have been synthesized in an attempt to obtain optimal potency, metabolic stability, receptor specificity, and favorable intravascular survival. Despite variations in these compounds, virtually of all of them retain the basic charge relations of the RGD sequence with a positive charge separated from a negative charge by approximately 10-20 .ANG. (Cook et al. ibid.; and Cox et al. ibid.).
Since .alpha..sub.v .beta..sub.3 is found on endothelial cells, and perhaps smooth muscle cells (Felding-Habermann et al. Curr. Opin. Cell Biol. 1993; 5:864-868), there are many potential sites of action. Recently Choi et al. demonstrated that a peptide that blocks .alpha..sub.v .beta..sub.3 prevented intimal hyperplasia after vascular injury in the rat (Choi et al. J. Vasc. Surg. 1994; 19:125-134), and Matsuno et al. demonstrated that a peptide that reacts with GPIIIb/IIIa and .alpha..sub.v .beta..sub.3 prevents neointima formation in the hamster (Matsuno et al. Circulation 1994; 90:2203-2206). Whether the peptide used by Choi et al. also inhibited rat platelet GPIIb/IIIa is not known.
Vitronectin (serum spreading factor or S protein) is a 75-kDa glycoprotein found in plasma (500 .mu.g/mL) and in extracellular matrix, including endothelial cell subendothelium (Barnes et al. J. Biol. Chem. 258; 12548 (1983); Hayman et al. Proc. Natl. Acad. Sci. U.S.A. 80; 4003, (1983); and Preissner et al. Blood 71; 1381 (1986)). Endothelial cells express a surface receptor for vitronectin and bind vitronectin (Fitzgerald et al. Biochemistry 26: 8158 (1987); Cheresh et al. Proc. Natl. Acad. Sci. USA 84; 6471 (1989); Cheng et al. J. Cell Physiol. 139; 275 (1989); Preissner et al. ibid.; and Polack et al. Blood 73; 1519 (1989)). Vitronectin mediates attachment and spreading of endothelial cells, the development of focal adhesion plaques, and clustering of the vitronectin receptor (Dejana et al. Blood 75; 1509 (1990); Dejana et al. J. Cell Biol. 107;1215 (1988); Dejana et al. Blood 71;566 (1988); Charo et al. J. Biol. Chem. 262;9935 (1987); Cheresh et al. Proc. Natl Acad. Sci. USA 84;6471, (1987); Cheng et al. J. Cell Physiol. 139;275 (1989); Barnes et al. J. Biol. Chem. 258:12548 (1983); Hayman et al. J. Cell Biol. 95;20 (1982)). Vitronectin is also found in platelets and is released when platelets are activated; vitronectin then binds to platelets, probably to GP IIIb-IIIa (Barnes et al. Proc. Natl. Acad. Sci. USA 80;1362 (1983)). Vitronectin thus acts as a subendothelial attachment factor for both endothelial cells and platelets. Vitronectin also mediates the adherence of group A and G streptococci to endothelial cells.
Compounds which are .alpha.v.beta.3 antagonists are useful for inhibiting bone resorption, treating and preventing osteoporosis, and inhibiting vascular restenosis, diabetic retinopathy, angiogenesis, artherosclerosis and tumor metastasis.
Osteoclasts are multinucleated cells of up to 400 .mu.m in diameter that resorb mineralized tissue, chiefly calcium carbonate and calcium phosphate, in vertebrates. They are actively motile cells that migrate along the surface of bone. They can bind to bone, secrete necessary acids and proteases and thereby cause the actual resorption of mineralized tissue from the bone.
More specifically, osteoclasts are believed to exist in at least two physiological states. In the secretory state, osteoclasts are flat, attach to the bone matrix via a tight attachment zone (sealing zone), become highly polarized, form a ruffled border, and secrete lysosomal enzymes and acids to resorb bone. The adhesion of osteoclasts to bone surfaces is an important initial step in bone resorption. In the migratory or motile state, the osteoclasts migrate across bone matrix and do not take part in resorption until they attach again to bone.
Integrins are transmembrane, heterodimeric, glycoproteins which interact with extracellular matrix and are involved in osteoclast attachment, activation and migration. The most abundant integrin in osteoclasts (rat, chicken, mouse and human) is the vitronectin receptor, or .alpha.v.beta.3, thought to interact in bone with matrix proteins that contain the RGD sequence. Antibodies to .alpha.v.beta.3 block bone resorption in vitro indicating that this integrin plays a key role in the resorptive process. There is increasing evidence to suggest that .alpha.v.beta.3 ligands can be used effectively to inhibit osteoclast mediated bone resorption in vivo in mammals.
The current major bone diseases of public concern are osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget's disease, immobilization-induced osteopenia, and glucocorticoid treatment.
Additionally, .alpha.v.beta.3 ligands have been found to be useful in treating and/or inhibiting restenosis (recurrence of stenosis after angioplasty or corrective surgery on the heart valve), artherosclerosis, diabetic retinopathy and angiogenesis (formation of new blood vessels). Moreover, it has been postulated that the growth of tumors depends on an adequate blood supply, which in turn is dependent on the growth of new vessels into the tumor; thus, inhibition of angiogenesis can cause tumor regression in animal models. (See, Harrison's Principles of Internal Medicine, 12th ed., 1991). .alpha.v.beta.3 antagonists, which inhibit angiogenesis, are therefore useful in the treatment of cancer for inhibiting tumor growth. (See e.g., Brooks et al., Cell, 79:1157-1164 (1994)).
Oral integrin receptor antagonists are readily absorbed when a patient consumes them on an empty stomach. However, it has been recently observed that absorption and bioavailability of oral integrin receptor antagonists, when taken with food, may be reduced by the presence of food in the stomach. The present compositions and methods provide a means for systemically delivering to a patient therapeutically effective amounts of integrin receptor antagonists.